Abstract

The germfree mouse model of Vibrio cholerae infection can be used to judge immune responses to V. cholerae vaccine and vector strains. In the original model, a single oral inoculation was administered on day 0, a booster oral inoculation was administered on day 14, and immune responses were analyzed with samples collected on day 28. Unfortunately, immune responses in this model frequently were low level, and interanimal variability occurred. In order to improve this model, we evaluated various primary and booster V. cholerae inoculation schedules. The most prominent systemic and mucosal antibody responses were measured in mice that received a multiple primary inoculation series on days 0, 2, 4, and 6 and booster inoculations on days 28 and 42. These modifications result in improved preliminary evaluation of V. cholerae vaccine and vector strains in mice.

Geometric mean titers (GMT) of vibriocidal antibody responses on day 28, 42, and 56 following oral inoculation of mice with V. cholerae vaccine strains by using various inoculation schedules (the fewest mice were evaluated on day 56): single inoculation (on day 56, the number of evaluated mice was 16), multiple inoculation I (on day 56, the number of evaluated mice was 15), and multiple inoculation II (on day 56, the number of evaluated mice was 9). Mice received either control strain Peru2ΔglnA(pKEK71-NotI) or vaccine strain Peru2ΔglnA(pTIC5). Within each inoculation group, no difference was detected between mice that received Peru2ΔglnA(pTIC5) or Peru2ΔglnA(pKEK71-NotI) (data not shown), and, therefore, mean vibriocidal-antibody titers are grouped by inoculation schedule. Error bars depict standard errors of the mean for each group. +, P ≤ 0.01 compared to animals receiving the single primary inoculation.

Serum IgG anti-CtxB ELISA results for day 28, 42, and 56 samples from mice inoculated with V. cholerae control strain Peru2ΔglnA(pKEK71-NotI) (hatched columns) or vaccine strain Peru2ΔglnA(pTIC5) (solid columns) by using various inoculation schedules as for Fig. 1 (the fewest mice were evaluated on day 56). On day 56, the number of evaluated control mice that had been inoculated with Peru2ΔglnA(pKEK71-NotI) in the single primary inoculation group was 7, in multiple primary inoculation group I it was 4, and in multiple primary inoculation group II it was 4. On day 56, the number of evaluated mice that had been inoculated with vaccine strain Peru2ΔglnA(pTIC5) in the single primary inoculation group was 9, in multiple primary inoculation group I it was 11, and in multiple primary inoculation group II it was 5. The geometric mean plus the standard error of the mean is reported for each group. mOD, milli-optical density units. #, P ≤ 0.001; ∗∗, P ≤ 0.02, compared to animals receiving vaccine strain Peru2ΔglnA(pTIC5) with the single primary inoculation schedule.

Serum IgA anti-CtxB ELISA results for day 28, 42, and 56 samples from mice inoculated with V. cholerae control strain Peru2ΔglnA(pKEK71-NotI) (hatched columns) or vaccine strain Peru2ΔglnA(pTIC5) (solid columns) using various inoculation schedules as for Fig. 1. For the number of mice in each group, see the legend to Fig. 2. The geometric mean plus the standard error of the mean is reported for each group. mOD, milli-optical density units. ∗∗, P ≤ 0.02, and ∗, P ≤ 0.05, compared to animals receiving vaccine strain Peru2ΔglnA(pTIC5) with the single primary inoculation schedule.

Bile IgA anti-CtxB ELISA results on day 56 samples from mice inoculated with V. cholerae control strain Peru2ΔglnA(pKEK71-NotI) (hatched columns) or vaccine strain Peru2ΔglnA(pTIC5) (solid columns) by using various inoculation schedules as for Fig. 1. For the number of mice in each group, see the legend to Fig. 2. The geometric mean plus the standard error of the mean is reported for each group. mOD, milli-optical density units. ∗, P ≤ 0.05 compared to animals receiving vaccine strain Peru2ΔglnA(pTIC5) with the single primary inoculation schedule.